This invention relates to a method, based on an immunological approach, for detecting the presence of a Mycobacterium species and a kit and antibodies for use therein.
The various known species of Mycobacterium are known to cause a number of infectious diseases in humans and animals. One such Mycobacterium species, Mycobacterium tuberculosis, causes tuberculosis in humans.
Tuberculosis is considered to be the major communicable disease throughout most of the world. Despite great advances in medical science and a range of effective drugs, which for some time created the impression that the disease had been conquered, and despite organised international efforts, tuberculosis remains a world health problem of staggering proportions. More than 8 million new cases world-wide and more than 3 million deaths were reported in the year 1990 alone (Snider, 1994). Predictions made by the World Health Organisation indicate that by the year 2000 the annual figures will grow to 10.2 million new cases and 3.5 million deaths, with Asia and Sub-Saharan Africa being the most affected continents (Dolin, Raviglione and Koch, 1994). The global distribution of the estimated number of tuberculosis cases for the current decade and the estimated number of deaths for the same period is presented in Dolin, Raviglione and Koch, 1994.
The close association documented between tuberculosis and AIDS as well as the frequently concomitant presence of both these diseases add gravity to the situation (Torres et al., 1990; De Cock, 1994; Cantwell and Binkin, 1994; Murray, 1994). The emergence of multiple-drug resistance among the strans of Mycobacterium tuberculosis and other atypical mycobacteria has introduced an additional dimension to this gigantic problem (Blumberg, Miller and Koornhof, 1994; Morse, 1994).
The accurate and timely detection of tuberculosis and related mycobacterial diseases is one of the important requirements to develop a more successful global strategy to combat these diseases.
Traditional laboratory detection methods have major disadvantages of either not being capable of distinguishing between live and dead bacilli (the quick and simple Ziehl-Neelsen staining) or, if these methods confirm the presence of the live bacilli (direct cultivation), a number of weeks is required before the laboratory tests are completed. This, in turn, may delay the commencement of treatment and may lead to further spread of the disease.
The more recent approaches are based either on the detection of patients"" response to the infection by methods such as serological tests, lymphocyte proliferative responses to mycobacterial antigens or by determining the level of adenosine deaminase, or on the detection of mycobacterial antigens and constituents using immunoassays such as ELISAs, gas and liquid chromatography or mass spectrophotometry. More modern, molecular approaches include polymerase chain reaction (PCR), DNA probes or DNA fingerprinting (Musial et al., 1988; Godfrey-Faussett, 1994).
Mason et al, 1993 (Tubercle and Lung Disease) describe a method of identification and characterization of mycobacterial species by using monoclonal antibodies inter alia directed to mycobacterial lipoarabinomannan and unspecified glycolipids.
Wiker et al, 1991 (Journal of General Microbiology) describe a method for characterizing and identifying mycobacterial species grown in culture by detecting characteristic protein antigens of mycobacteria.
Hamid et al, 1993 (Journal of General Biology) describe a text to distinguish Mycobacteria from other bacteria by precipitating mycolic acids and performing a TLC analysis on them.
Young 1980 (Journal of General Biology) describe a method for extracting and analyzing mycolic acids from mycobacteria by TLC and shows a difference in the mycolic acid profile between Mycobacterium leprae and other bacteria in leprosy biopsies.
European Patent Application No 0407605 details a method of detecting antibodies in patient sera to bacteria from the species Nocardia by detecting antibodies raised to nocardial mycolic acids.
However, none of the methods listed above fulfills all the requirements for a quick, simple and reliable test capable of distinguishing between live and dead mycobacteria cells.
According to the invention there is provided a diagnostic assay for detecting and identifying a Mycobacterium species in a biological sample of an animal by immunologically detecting at least one intra-cellular mycobacterial antigen of the species in. the sample.
xe2x80x9cIntra-cellular mycobacterial antigenxe2x80x9d is defined herein to be a non-surface antigen or a metabolic product of the Mycobacterium species.
The intra-cellular mycobacterial antigen is preferably detected by exposing the biological sample to an antibody or antibodies specific for the intra-cellular mycobacterial antigen.
The assay preferably comprises the step of lysing at least some of the mycobacterial cells present in the sample to release at least some of the antigen present in their cell walls prior to contacting the sample with antibody.
The assay preferably also comprises the steps of treating the biological sample to release the mycobacterial cells from any organic debris in which they may be embedded and decontaminating the biological sample to eliminate unwanted microorganisms present in it prior to lysing the mycobacterial cells.
The intra-cellular mycobacterial antigen is preferably a mixture of mycolic acids present in the cell wall of the Mycobacterium species, each having the following general structure: 
The biological sample is preferably sputum, blood, cerebro-spinal fluid, stool, urine, gastric lavage, saliva, tissue, a laryngeal swab or an exudate from a skin lesion.
The assay preferably also comprises the step of concentrating the mycolic acids in the sample by an immobilized antibody affinity procedure prior to contacting them with a labelled antibody. The concentration step is preferably carried out using an immuno-affinity column.
According to another aspect of the invention a method for group separation and subsequent purification of mycobacterial mycolic acids having the general structure set out above from a mixture of extracted mycobacterial mycolic acids and contaminants comprises the steps of:
dissolving the mixture of mycolic acids and contaminants in a bi-phasic solvent; and
subjecting the mixture to liquid-liquid phase separation.
Preferably, the purified mycolic acids are not subsequently chemically derivatized.
The bi-phasic solvent system preferably comprises chloroform, methanol and water.
Preferably, the bi-phasic solvent system comprises an upper phase and a lower phase.
The method preferably also comprises the steps of mixing and equilibrating the upper and lower phases.
Preferably, the composition of the upper phase is 12-18% chloroform, 45-55% methanol and 20-40% water. More preferably, the composition of the upper phase is 15% chloroform, 52% methanol and 33% water.
Preferably, the composition of the lower phase is 50-80% chloroform, 5-40% methanol and 2-8% water. More preferably, the composition of the lower phase is 68% chloroform, 27% methanol and 5% water.
The purification is preferably performed using a countercurrent apparatus or any other liquid-liquid extractor.
According to another aspect of the invention a purified mycolic acid having the following general structure: 
group separated and purified is provided.
According to another aspect of the invention an antibody to an intra-cellular mycobacterial antigen is provided.
The antibody may be monoclonal or polyclonal and is preferably of animal origin.
The antibody is preferably an isolated polyclonal antibody raised to a mycolic acid purified by the above method.
According to another aspect of the invention a mycobacterial intra-cellular antigen/carrier conjugate is provided.
The intra-cellular antigen is preferably a mycolic acid and it is preferably adsorbed onto the carrier.
The carrier may be a protein such as bovine serum albumin, gelatin or keyhole limpets hemocyanin.
According to yet another aspect of the invention a kit for detecting the presence of a Mycobacterium species in a biological sample comprises:
an antibody raised to a mycolic acid purified by the above method and detectably labelled.
The kit may also comprise an amount of a mycobacterial mycolic acid/carrier conjugate and optionally also an immobilized antibody.